Peripheral neuropathies and nerve injuries are common clinical problems, and the only medical therapy is to identify and eliminate their cause. Since axonal regeneration occurs in peripheral nerves, and is a major mechanism by which recovery occurs in patients who have a neuropathy or nerve injury, we need to understand axonal regeneration at a molecular level in order to create new therapies. Schwann cells play a central role in axonal regeneration. When Schwann cells are 'denervated' by the loss of axonal contact, they undergo profound changes that are part of what is termed Wallerian degeneration. In neuropathies and nerve injuries, these denervated Schwann cells and their basal laminae provide the pathway through which axons regenerate. To understand how Schwann cells promote axonal regeneration at a molecular level, we need to learn what genes are up- and down-regulated, what roles these genes play in Wallerian degeneration, and how they are regulated. The approach will be to make a cDNA library from degenerating rat sciatic nerve, and screen this library with cDNA probes from normal sciatic nerve and from degenerating sciatic nerve, thereby identifying clones that are differentially expressed in degenerating nerve. Clones that are up- regulated and down-regulated during Wallerian degeneration will be collected, and the former will be analysed first, as these seem more likely to play a role in axonal regeneration. Clones representing less abundant cDNAs that are up-regulating during Wallerian degeneration will be isolated with a subtractive probe. Clones will be characterized by (a) cross- hybridization to find the ones that are copies of the same gene, (b) by performing Northern blots to confirm that the clones are truly up-regulated during Wallerian degeneration and to determine the size of their mRNA, and (c) by partially sequencing clones to see if they are novel or have previously been described. Novel clones that are up-regulated will be completely sequenced, their amino acid sequence will be deduced, and their cellular localization will be demonstrated by in situ hybridization & immunohistochemistry.
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